Validation of reference gene stability for miRNA quantification by reverse transcription quantitative PCR in the peripheral blood of patients with COVID-19 critical illness.

The COVID-19 pandemic has created an urgency to study the host gene response that leads to variable clinical presentations of the disease, particularly the critical illness response. miRNAs have been implicated in the mechanism of host immune dysregulation and thus hold potential as biomarkers and/or therapeutic agents with clinical application. Hence, further analyses of their altered expression in COVID-19 is warranted. An important basis for this is identifying appropriate reference genes for high quality expression analysis studies. In the current report, NanoString technology was used to study the expression of 798 miRNAs in the peripheral blood of 24 critically ill patients, 12 had COVID-19 and 12 were COVID-19 negative. A list of potentially stable candidate reference genes was generated that included ten miRNAs. The top six were analyzed using reverse transcription quantitative polymerase chain reaction (RT-qPCR) in a total of 41 patients so as to apply standard computational algorithms for validating reference genes, namely geNorm, NormFinder, BestKeeper and RefFinder. There was general agreement among all four algorithms in the ranking of four stable miRNAs: miR-186-5p, miR-148b-3p, miR-194-5p and miR-448. A detailed analysis of their output rankings led to the conclusion that miR-186-5p and miR-148b-3p are appropriate reference genes for miRNA expression studies using PaxGene tubes in the peripheral blood of patients critically ill with COVID-19 disease.

Effects of the vasopeptidase inhibitor omapatrilat on peri- and postmyocardial infarction in Zucker lean rats.

BACKGROUND: The vasopeptidase inhibitor omapatrilat improves insulin sensitivity and survival following myocardial infarction (MI). It also improves left ventricular (LV) remodelling following MI and reduces MI size. OBJECTIVES: To determine whether improvement in LV remodelling and function is accompanied by a reduction in fetal gene expression of the contractile apparatus, and whether reduction in MI size is accompanied by an increase in the expression of the glucose transporter GLUT-4. METHODS: Eighty-nine rats were pretreated for seven days with omapatrilat 20 mg/kg/day and 91 rats were left untreated. MI was induced in 180 Zucker lean male rats by ligating the left anterior descending coronary artery, and omapatrilat was given for another 38 days in the survivors. After 30 days, echocardiography was performed. At 38 days, hemodynamic measurements were performed, the rats were sacrificed and morphological measurements were done. Using quantitative reverse transcriptase-polymerase chain reaction, gene expression was measured in the LV using transcript levels. RESULTS: Treatment with omapatrilat resulted in improved early (24 h) and late (38 days) survival following MI (50% to 67%, P=0.023, and 44% to 59%, P=0.045, respectively). Omapatrilat treatment reduced MI size and resulted in beneficial ventricular remodelling as reflected by a reduction in cardiac dimensions by echocardiography, and LV and right ventricular hypertrophy, which resulted in borderline hemodynamic improvement. A large MI resulted in an increased expression of beta-myosin heavy chain, alpha-skeletal actin and atrial natriuretic peptide, and a decreased expression of GLUT-4. Omapatrilat treatment did not modify the expression of these genes. CONCLUSIONS: The results suggest that the vasopeptidase inhibitor omapatrilat does not modify fetal gene expression of the contractile apparatus or the expression of GLUT-4 despite reducing cardiac hypertrophy and MI size.